1. Field of the Invention
The invention concerns a microstructured, planar biosensor, in particular a biosensor with an enzyme as a biocatalyst. The invention also concerns the use of the biosensor and a process for immobilizing biocatalysts.
Analytical devices are already known with detection layers of enzymatic membranes or enzyme layers (both terms being used synonymously here), through which the change in hydrogen peroxide or oxygen concentration resulting from an enzymatic turnover, e.g. of oxygen, can be determined electrochemically or amperometrically (F. Scheller et al., xe2x80x9cEnzym- und Zellsensorenxe2x80x94Anwendungen, Trends und Perspektivenxe2x80x9d, Spektrum der Wissenschaft, September 1992, pages 99 to 103). The functionality, for example the saturation characteristics and sensitivity, of such a biosensor depends primarily on the quality of the enzymatic membrane used. A number of criteria must be met in fabricating the membrane (P. Vadgama and P. W. Crump, titled xe2x80x9cBiosensors: Recent Trendsxe2x80x9d, Analyst, November 1992, Vol. 117, pages 1657 to 1670). The maximum possible amount of active enzyme must be immobilized or bound in such a way that it largely remains functional and active while, at the same time, being attached to the membrane. A number of techniques have been developed for achieving this binding of the enzyme in the membrane in as gentle a way as possible. The following methods are used: adsorption, ionic binding, absorption, enclosure in microcapsules or in membranes, and covalent binding to carrier substances.
On the one hand, fabrication of the enzymatic membrane takes place through the enzyme being physically bound in polymeric xe2x80x9cgelsxe2x80x9d, e.g. poly(hydroxyethyl methacrylate), in which the enzyme is retained as if in nets. This method suffers the disadvantage that interactions always arise between the gel molecules and the enzymatic proteins, which affect the flexibility of the three-dimensional structure and thereby negatively influence the activity of the enzymes. Alternatively, techniques are used through which the enzymatic proteins are attached directly to polymeric carriers over covalent chemical bonds. In this case, of course, the dynamics of the tertiary structure of the protein is influenced to an even greater extent by cross-linking between the amino-acid side-chains of the enzyme and the active groups of the carrier. Since, however, this structure is an important requirement for the activity of the enzyme, such a restriction of the structural flexibility is associated with partial inactivation of the enzyme.
European Patent EP 0 588 153 B1 discloses a structure of a planar, microstructured gas sensor chip with a first, inner, and a second, outer, limiting structure. This structure enables the formation of a pot and lid structure for a measuring solution (electrolyte solution) in that the electrolyte solution is placed in the first, inner limiting structure (pot) and a hydrophobic and protective material (lid) is placed in the second, outer limiting structure in such a way that it completely covers the electrolyte solution together with the first, inner limiting structure. This planar sensor chip is used for the determination of electrochemical gases such as oxygen and carbon dioxide.
Up to now there has been no pot and lid structure for biosensor determinations with a sensor chip of this kind because only the enzymatic membrane, which is formed as a single homogeneous layer, was available as a detection layer (xe2x80x9cmeasuring solutionxe2x80x9d). Thus up now, the two limiting structures could not be exploited for determinations using biosensors because formation of the pot and lid structure requires the existence of two layers instead of a single homogeneous layer.
It is accordingly an object of the invention to provide a microstructured biosensor, use of the biosensor and a process for the immobilization of biocatalysts that overcome the above-mentioned disadvantages of the prior art devices and methods of this general type, which has improved functionality, whose detection layer consists of two layers and in which the dynamics of the tertiary structure of the enzymes remains unchanged. Finally, the object of the invention is to provide a process with which a pot and lid structure can be fabricated on a sensor chip with an inner and an outer limiting structure.
With the foregoing and other objects in view there is provided, in accordance with the invention, a microstructured, planar biosensor, in which a biomolecular turnover to be measured can be determined electrochemically, the biosensor including:
a planar substrate;
a first, inner limiting structure defining a first interior space disposed on the planar substrate;
an enzymatic layer having an enzyme in a crystalline form disposed in the first interior space;
a second, outer limiting structure defining a second interior space disposed on the planar substrate; and
a polymeric hydrophilic covering layer disposed in the second interior space completely covering the first, inner limiting structure containing the enzymatic layer.
The subject of the invention is therefore a microstructured, planar biosensor with which the biomolecular turnover to be measured is determined electrochemically, whereby the sensor contains at least two limiting structures on a planar substrate. A first, inner limiting structure filled with an enzymatic membrane, and a second, outer limiting structure filled with a polymeric covering layer completely covering the first limiting structure with the enzymatic membrane is provided.
A further subject of the invention is the use of the biosensor in medicine, namely for the minimally invasive determination of principal metabolites in blood, such as glucose and lactate.
The final subject of the invention is a process for the immobilization of biocatalysts in which, in a first step, the biocatalyst in a liquid solution is filled onto a substrate in a first, inner limiting structure, is dried there in a second step, and is finally coated with a polymerizable covering layer in a third step.
Other features which are considered as characteristic for the invention are set forth in the appended claims.
Although the invention is illustrated and described herein as embodied in a microstructured biosensor, use of the biosensor and a process for the immobilization of biocatalysts, it is nevertheless not intended to be limited to the details shown, since various modifications and structural changes may be made therein without departing from the spirit of the invention and within the scope and range of equivalents of the claims.